Thrombosis Research (2007) 120, 323–336

intl.elsevierhealth.com/journals/thre

REVIEW ARTICLE

Measuring antiplatelet drug effects

in the laboratory
Paul Harrison a , A.L. Frelinger III b,d ,
Mark I. Furman b,c,e , Alan D. Michelson b,d,e,⁎

a
Oxford Haemophilia Centre and Thrombosis Unit, Churchill Hospital, Oxford, United Kingdom
b
Center for Platelet Function Studies, University of Massachusetts Medical School, Worcester, MA, USA
c
Division of Cardiovascular Medicine, University of Massachusetts Medical School, Worcester, MA, USA
d
Department of Pediatrics, University of Massachusetts Medical School, Worcester, MA, USA
e
Department of Medicine, University of Massachusetts Medical School, Worcester, MA, USA

Received 14 October 2006; received in revised form 14 October 2006; accepted 27 November 2006
Available online 17 January 2007

Abstract This review discusses the advantages and disadvantages of currently

available tests for the monitoring of antiplatelet therapy (especially aspirin and
clopidogrel). Many tests of platelet function are now available for clinical use, and some
of these tests have been shown to predict clinical outcomes after antiplatelet therapy.
However, in most of these studies, the number of major adverse clinical events was low.
No published studies address the clinical effectiveness of altering therapy based on the
results of monitoring antiplatelet therapy. Therefore, the correct treatment, if any, of
"resistance" to antiplatelet therapy is unknown and, other than in research trials,
monitoring of antiplatelet therapy in patients is not generally recommended. A clinically
meaningful definition of "resistance" to antiplatelet drugs needs to be developed, based
on data linking drug-dependent laboratory tests to clinical outcomes in patients.
© 2006 Elsevier Ltd. All rights reserved.

Contents

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 324
History of platelet function testing and overview of currently available tests . . . . . . . . . . . . . . 324
Monitoring of anti-platelet therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
Monitoring of aspirin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 328
Monitoring of clopidogrel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 330
Monitoring of GPIIb–IIIa antagonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 331

⁎ Corresponding author. Center for Platelet Function Studies, University of Massachusetts Medical School, Room S5-846, 55 Lake
Avenue North, Worcester, MA 01655, USA. Tel.: +1 508 856 0056; fax: +1 508 856 4282.
E-mail address: michelson@platelets.org (A.D. Michelson).

0049-3848/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.thromres.2006.11.012
324
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Introduction
Most platelet function tests have been traditionally
utilized for the diagnosis and management of
patients presenting with bleeding problems rather
than thrombosis [1]. However, as platelets are now
implicated in the development of atherothrombosis,
which is the leading cause of mortality in the
Western world,[2,3] new and existing platelet
function tests are increasingly being used for
monitoring the efficacy of the antiplatelet drugs
used to treat these conditions. This, coupled with
the development of new, simpler tests and point-of-
care (POC) instruments, has resulted in the increas-
ing tendency of platelet function testing to be
performed away from specialized hemostasis clini-
cal or research laboratories, where the more
traditional and complex tests are still performed
[4,5]. Table 1 is a summary of the currently available
tests for the monitoring of antiplatelet therapy,
including their advantages and disadvantages.
History of platelet function testing and
overview of currently available tests
Platelets were discovered in the 1880's [6]. Platelet
function testing began with the application of the in
vivo bleeding time by Duke in 1910 [7]. The bleeding
time was still regarded as the most useful screening
test of platelet function until the early 1990's [1,8,9].
Recently, the widespread use of the bleeding time has
rapidly declined because its limitations have been
recognized (see below) and other, less invasive,
screening tests have become available [10–12].
Light transmission aggregometry (LTA) was
invented in the 1960's and soon revolutionized the
diagnosis of primary hemostatic defects [13,14]. LTA
is still regarded as the gold standard of platelet
function testing and by adding a panel of agonists to
stirred platelets it is possible to obtain a large
amount of information about many different
aspects of platelet function [15]. This test, often
now coupled with the measurement of stored and
releasable platelet nucleotide content, is still
utilized in most laboratories for the diagnosis of
many platelet defects [16]. Recently, commercial
aggregometers have become easier to use with
multi-channel capability, simple automatic setting
of 100% and 0% baselines, and computer operation
and storage of results. For example, a new fully
computerized 8-channel aggregometer has just
P. Harrison et al.
332
332
become available (Fig. 1). Some instruments can
simultaneously measure luminescence, to monitor
the release reaction of dense granular nucleotides
during secondary aggregation. LTA is relatively non-
physiological, as separated platelets are stirred
under low shear conditions and only form aggre-
gates after addition of agonists, conditions which do
not accurately mimic platelet adhesion, activation
and aggregation upon vessel wall damage. Also,
conventional LTA using a full panel of agonists
requires both large blood volumes and a significant
expertise to perform the tests and interpret the
tracings. In response to the problems with the
bleeding time and LTA, a number of alternative tests
have been developed, including impedance whole
blood aggregometry (WBA), a fully automated
cartridge-based instrument (VerifyNow®) that mea-
sures platelet LTA in anticoagulated whole blood,
and a variety of tests that attempt to simulate
primary hemostasis in vitro (Table 1).
WBA provides a means to study platelet function in
anticoagulated whole blood [17]. The test measures
the change in resistance or impedance between two
electrodes as platelets adhere and aggregate in
response to classical agonists. A new fully computer-
ized two or four channel instrument has now become
available (Fig. 2). Although the latter instrument can
also be used for LTA of platelet-rich plasma (PRP),
WBA has many significant advantages including the
use of smaller sample volumes and the immediate
analysis of samples without manipulation, loss of time
or potential loss of platelet subpopulations or
platelet activation during centrifugation. A new five
channel computerized WBA instrument (Multiple
Platelet Function Analyzer or Multiplate®) now has
disposable cuvettes/electrodes with a range of
different agonists for both diagnosis and monitoring
antiplatelet therapy.
The VerifyNow® (formerly known as the Ultegra
Rapid Platelet Function Analyzer [RPFA]) instrument
(Fig. 3) is a fully automated POC test that was
originally developed to monitor glycoprotein (GP)
IIb–IIIa antagonists within a self-contained cartridge
(containing a platelet activator and fibrinogen-
coated beads) [18–20]. Blood sample tubes are then
simply mixed prior to insertion onto the cartridge
that has been pre-mounted onto the instrument.
Aggregation in response to the agonist is monitored
by light transmission through two duplicate reaction
chambers in each cartridge. Other specialized car-
tridges are now available for measuring platelet
 Measuring antiplatelet drug effects in the laboratory
Table 1 An alphabetical list of currently available tests for the monitoring of antiplatelet therapy
Name of test Principle Advantages Disadvantages Frequency of use
AspirinWorks® Immunoassay of urinary Measures stable Indirect assay Increasing use
11-dehydrothromboxane B2 thromboxane metabolite Not platelet-specific
Dependent upon COX-1 activity Renal function-dependent

Bleeding time In vivo cessation of blood flow In vivo test Insensitive Decreasing popularity
Physiological POC Invasive
Scarring
High CV
Flow Cytometry Measurement of platelet glycoproteins and Whole blood test Specialized operator Widely used
activation markers by fluorescence (e.g. VASP Small blood volumes Expensive
phosphorylation to monitor P2Y12 inhibition) Wide variety of tests
HemoStatus® Device Platelet procoagulant activity Simple Insensitive to aspirin and GPIb function Used in surgery and cardiology
POC
Ichor–Plateletworks® Platelet counting pre- and post-activation Rapid Indirect test measuring count after Used in surgery and cardiology
Simple aggregation
POC
Small blood volume
Impact® cone and Quantification of high shear platelet Small blood volume required Instrument not yet widely available. Little widespread experience as only
plate(let) analyzer adhesion/aggregation onto surface High shear recently commercially available
Rapid
Simple
Research (variable) and fixed
versions available POC
Light Transmission Low shear platelet-to-platelet aggregation Historical gold standard Time consuming Widely used in specialized labs
Aggregometry (LTA) in response to classical agonists Sample preparation
Expensive
PFA-100® High shear platelet adhesion and aggregation Whole blood test Inflexible Widely used
during formation of a platelet plug High shear VWF-dependent
Small blood volumes Hct-dependent
Simple Insensitive to clopidogrel
Rapid
POC
Platelet Reactivity Measurement of platelet aggregates in whole Simple Requires blood counter Little widespread experience
Index blood (modified Wu and Hoak method) Rapid Indirect test measuring count after aggregation
Inexpensive
Serum Thromboxane Immunoassay Dependent upon COX-1 activity Prone to artefact Widespread use
B2 Not platelet-specific
Thromboelastography Monitoring of rate and quality of clot Global whole blood test POC Measures clot properties only; largely platelet-independent Used in surgery and anesthesiology
(TEG® or ROTEM®) formation unless platelet activators are used
VerifyNow® Fully automated platelet aggregometer to Simple Inflexible Increasing use
measure antiplatelet therapy POC 3 test cartridges (aspirin, Cartridges can only be used for single purpose
P2Y12 and GPIIb–IIIa)
Whole Blood Monitors changes in impedance in response Whole blood test Older instruments require electrodes to be Widely used in specialized labs although
Aggregometry to classical agonists cleaned and recycled less than LTA
Abbreviations: COX-1, cyclooxygenase 1; CV, coefficient of variation; GP, glycoprotein; Hct, hematocrit; LTA, light transmission aggregometry; PFA-100, platelet function analyzer 100; POC, point-of-care; VASP,
vasodilator-stimulated phosphoprotein; VWF, von Willebrand factor.

325
326 P. Harrison et al.

responses to either aspirin (VerifyNow® Aspirin) or

clopidogrel and other P2Y12 antagonists (VerifyNow®
P2Y12). This instrument is a considerable advance, as
the test is a fully automated POC test without the
requirements of sample transport, time delays or a
specialized laboratory, and it can provide immediate
information.
It is also possible to monitor platelet aggregometry
in whole blood by a simple platelet counting tech-
nique. After addition of an agonist to anticoagulated,
stirred whole blood, platelets aggregate and the
platelet count decreases when compared to a control
tube [21–23]. The Plateletworks® aggregation kits
(Helena Biosciences) are simply based upon comparing
platelet counts within a control EDTA tube and after
aggregation with either ADP or collagen within
citrated tubes [24-27].
A number of tests have also been developed that
attempted to mimic the processes that occur during Figure 2 The Chrono-log Model 700 whole blood/optical 2
vessel wall damage. Many of these techniques have channel lumiaggregometer. Reproduced with permission
from Chrono-log.
remained primarily research tools within expert
laboratories because of their inherent complexity
and technical difficulty. Commercially available collagen or extracellular matrix (ECM) under high
instruments include the platelet function analyzer shear conditions of 1800 s − 1 [28–30]. In the
100 (PFA-100®, Fig. 4) and the Impact® cone and commercial version of the device, the Impact®
plate(let) analyzer (Fig. 5). Both of these tests (DiaMed), a plastic plate is utilized instead of a
measure platelet adhesion and aggregation under collagen or an ECM-coated surface. The test is now
conditions of high shear, in an attempt to simulate fully automated, simple to operate, uses a small
primary hemostatic mechanisms that are encoun- quantity of blood (0.12 mL) and displays results in
tered in vivo. 6 min. The instrument contains a microscope and
The cone and plate(let) analyzer, originally performs staining and image analysis of the plate-
developed by Varon and Savion, monitors platelet lets that have adhered and aggregated on the plate.
adhesion and aggregation to a plate coated with Preliminary data suggest the test can be used in the
diagnosis of platelet defects and monitoring anti-
platelet therapy. Because the test has only just
become commercially available, widespread expe-
rience is limited.
The PFA-100® device (Dade-Behring) has been
available for a number of years and is now in
widespread use within many laboratories, with over

Figure 1 An example of a modern 8-channel platelet

aggregometer. The model shown is the Biodata PAP-8E. Figure 3 The VerifyNow system. Reproduced with permis-
Reproduced with permission from Biodata and Biodis. sion from Accumetrics.
Measuring antiplatelet drug effects in the laboratory 327

computer. The cup oscillates 5° in each direction. In

normal anticoagulated blood the pin is unaffected,
but as the blood clots, the motion of the cup is
transmitted to the pin. Whole blood or re-calcified
plasma can be used, with or without activators of the
tissue factor or contact factor pathways. The TEG
provides a relatively rapid result (b 30 min), can be
conducted in a POC fashion and provides various
data relating to clot formation and lysis (the lag time
before the clot starts to form, the rate at which
clotting occurs, the maximal amplitude of the trace
and the extent and rate of amplitude reduction).
Rotational TEG (ROTEG® or ROTEM®) is an adapta-
tion of the TEG in which the cup is stationary and the
pin oscillates [42,45]. Unlike platelet function tests,
TEG instruments have been traditionally utilized
Figure 4 The PFA-100 instrument. Reproduced with per- within surgical and anesthesiology departments as
mission from Dade-Behring. POC tests for determining the risk of bleeding and as
a guide to transfusion requirements. More recent
200 papers published on various clinical applications developments include an expansion in the range of
[31,32]. The test was originally developed as a activators to initiate aggregation rather than coag-
prototype instrument called the Thrombostat 4000 ulation e.g. the platelet mapping system® using ADP
by Kratzer and Born [33,34]. The PFA-100® mea- and arachidonic acid [46,47]. The Haemostasis
sures the fall in flow rate as platelets within citrated Analysis System® (HAS) by Hemodyne is based upon
whole blood are aspirated through a capillary and the original technique developed by Carr [48–51].
begin to seal a 150 μm aperture within a collagen- The HAS® measures a number of parameters in
coated membrane. This reaction takes place clotting blood including platelet contractile force
contained within one of two types of disposable (PCF), clot elastic modulus and thrombin generation
cartridge (collagen–epinephrine or collagen–ADP). time (TGT) in a small sample (700 μL) of whole
The instrument records the time (closure time or blood.
CT) it takes to occlude the aperture, along with the
total volume of blood used during the test.
Platelets contribute significantly to the genera-
tion of thrombin and the dynamics of blood clotting
including clot formation, clot retraction and lysis.
Clot retraction can be easily measured in whole
blood or PRP within glass tubes after the addition of
calcium. The role of platelets in clot retraction was
first described by Hayem in the late 19th century and
Glanzmann famously described patients with poor
clot retraction or thrombasthenia in 1918, who were
subsequently shown to be defective in GPIIb–IIIa
[35]. Modern tests are available that can study both
the role of platelets in thrombin generation, clot
formation and clot retraction. For example, throm-
bin generation tests can be used to measure
thrombin generation in PRP and whole blood [36–
38]. The HemoStatus® test (Medtronic Blood Man-
agement, Parker, CO, USA) can be used to detect the
effects of GPIIb–IIIa antagonists [39–41]. There are
also a number of instruments that measure the
physical properties of clot formation. Thromboelas-
tography® (TEG) was developed more than 50 years
ago [42–44]. Anticoagulated whole blood is incu-
bated in a heated sample cup in which a pin is Figure 5 The Impact cone and plate(let) analyzer. Repro-
suspended that is connected to a chart recorder or duced with permission from DiaMed.
328
In the last 20 years, flow cytometric analysis of
platelets has also developed into a popular means to
study many aspects of platelet biology and function.
Preferred modern methods now utilize diluted
anticoagulated whole blood incubated with a
variety of reagents including antibodies and dyes
that bind specifically to individual platelet proteins,
granules and lipid membranes [52–54]. Flow cyto-
metric analysis of platelet function is discussed in
detail in Refs. [52–54].
Monitoring of anti-platelet therapy
Most platelet function tests have been traditionally
utilized to either screen for or diagnose platelet
defects. Most traditional tests are not only difficult
to perform but are expensive, time consuming, and
require relatively large volumes of fresh blood.
They are therefore usually performed within spe-
cialized hemostasis laboratories, often in close
proximity to associated clinics. Many of these tests
are limited in their capacity to predict bleeding or
thrombosis. These limitations have largely restrict-
ed their widespread clinical use within other
disciplines (e.g., cardiology, stroke and surgery).
However, this is now beginning to change as simpler
tests of platelet function become available that can
potentially be utilized as POC tests or at least within
non-specialized laboratories. With the increasing
development of new classes of antiplatelet drugs
and the known heterogeneity in their biological
effects between patients, it may become useful to
monitor an individual's response to antiplatelet
therapy so that either the dosage and/or the type
of drug(s) administered can be titrated or optimized
within individual patients to help control and
minimize the risk of either thrombosis or bleeding.
The antiplatelet drug aspirin has traditionally
been administered at a standard dose with no
monitoring of effect, on the assumption that usual
doses are two to three times those required to
inhibit all cyclooxygenase-1 (COX-1) activity. How-
ever, the lack of a simple, convenient, reliable and
clinically relevant test of platelet function has
meant that lack of effect in individual patients has
gone undetected. With the availability of other
classes of antiplatelet drugs (e.g. thienopyridines,
new P2Y12 antagonists and GPIIb–IIIa antagonists)
there is renewed interest in the potential utility of
platelet function tests to monitor the efficacy of
platelet inhibition. The development of GPIIb–IIIa
antagonists in particular resulted in the develop-
ment of a number of new assays to monitor a
patient's response (e.g., VerifyNow® IIb/IIIa) mainly
because of their narrow therapeutic window with
associated increased risk of bleeding. The still
P. Harrison et al.
poorly-defined phenomenon of “drug resistance”
has led to an explosion of interest, research and
availability of a variety of tests that can potentially
monitor an individual's response to antiplatelet
therapy [55]. The question remains as to whether
these tests will be clinically useful in the prediction
of bleeding and/or thrombosis. Patient non-compli-
ance to their therapy is also an important but
relatively common confounding problem in many
studies [55].
It is well known that there is considerable
variation in the response of individuals (either
patients or normal controls) to aspirin, clopidogrel
and GPIIb–IIIa antagonists as measured by various
platelet function tests. Those individuals who
respond poorly to a given drug are therefore termed
“resistant”. However, this is a poorly-defined
phenomenon and a precise definition of resistance
should only relate to the action of a specific drug to
inhibit its biochemical target [56]. Many platelet
function tests are non-specific and do not meet this
criterion. Resistance may simply represent natural
biological variation in a given drug response or may
be due to specific or more complicated mechanisms
[57]. Is resistance specific to an individual class of
drug related to its mechanism of action, or are there
common inherited and/or acquired mechanisms
that may influence an individual's response to not
just one but potentially all antiplatelet drugs [57]?
Recent data indeed suggests that aspirin resistant
patients as a group also have a reduced response to
clopidogrel [58]. Whatever the mechanisms, the key
question is whether a laboratory test that detects
resistance, or non-response, predicts future clinical
events — and whether changing therapy based on
the test is beneficial to the patient. Until these links
are firmly proven within large trials, resistance in
the laboratory cannot necessarily be ascribed as a
cause of thrombosis. Therefore, except in research
trials, it is still not yet clinically useful to test for
resistance and change a patient's therapy on the
basis of a laboratory test [55,57,59]. The following
sections discuss the specific laboratory tests for the
three main antiplatelet drugs.
Monitoring of aspirin
Aspirin irreversibly inhibits COX-1 resulting in the
inhibition of thromboxane (TX) A2 generation for the
entire lifespan of the platelet [60]. Aspirin is an
effective antiplatelet agent because it reduces the
relative risk of major vascular events and vascular
death by about 25% after ischemic stroke and acute
coronary syndrome [61]. Regular low doses of
aspirin (e.g., 81 mg/day) will usually result in
N 95% inhibition of thromboxane generation.
Measuring antiplatelet drug effects in the laboratory
Therapeutic monitoring was therefore considered
to be unnecessary. However, the antiplatelet
properties of aspirin have been shown to vary
between individuals and recurrent events in some
patients could be due to “aspirin resistance” or
aspirin non-responsiveness [56,57]. The reported
incidence of aspirin non-responsiveness varies
widely (between 5–60%). There are also many
possible mechanisms for aspirin resistance which
have been discussed in detail elsewhere [57,62].
Recently it has been proposed that the term “aspirin
resistance” should only be utilized as a description
of the failure of aspirin to inhibit TXA2 production,
irrespective of a non-specific test of platelet
function [56]. This is because there are many
other biochemical pathways that can potentially
bypass COX-1 even if this enzyme is inhibited.
Depending upon the test system employed, “aspirin
resistance” may therefore be detected even if COX-
1 is fully blocked [56]. Recent studies also suggest
that, in compliant patients, the incidence of aspirin
resistance is rare using methods dependent on COX-
1 activity [46,63,64]. Addition of in vitro aspirin to
samples followed by retesting should also be an
important consideration for testing compliance
[64,65].
Many tests have been used to assess the influence
of aspirin on platelets and aspirin resistance,
including arachidonic acid- and ADP-induced LTA,
ADP- and collagen-induced impedance aggregation,
VerifyNow® Aspirin, PFA-100®, Thromboelastogra-
phy® (TEG — platelet mapping system®), flow
cytometry using arachidonic acid stimulation, Im-
pact cone and plate(let) analyzer, and serum and
urinary thromboxane [55]. Tests should ideally be
performed pre- and post-drug. Some of the tests
have been reported to be predictive of adverse
clinical events [55]. However, the large majority of
these studies are small and often statistically
underpowered to completely answer whether each
test can reliably predict the small number of
adverse outcomes that were observed [57,62].
Although preliminary results from some studies
could suggest that responses to aspirin should be
monitored, there are additional problems in that
LTA is time-consuming, difficult and cannot realis-
tically be performed on large numbers of patients in
routine practice. However, the simpler tests of
platelet function (e.g. PFA-100®, VerifyNow® Aspi-
rin, TEG platelet mapping®, Impact®, and urinary
thromboxane) could offer the possibility of rapid
and reliable identification of aspirin non-responsive
patients. The PFA-100® usually gives a prolongation
in the Collagen/Epinephrine (CEPI) CT in response to
aspirin, with the Collagen/ADP (CADP) CT usually
remaining within the normal range [66,67]. A
329
number of studies have observed that an apprecia-
ble number of both normals and patients fail to
respond in terms of prolongation of their CEPI CT in
response to aspirin [68–75]. Because the PFA-100®
is a global high-shear test of platelet function, many
variables have been shown to influence the CT
including VWF levels, platelet count and hematocrit
[32]. In patients identified with “aspirin resistance”
by the PFA-100®, a number of studies have shown
that VWF levels are elevated in non-responders
[73,74,76]. As the CEPI CT is highly dependent upon
VWF and other variables, pre- and post-aspirin CTs
should ideally therefore be determined, because
the true aspirin response may be masked before the
drug is given [56]. Also, CADP CTs are lower in these
patients, which may be caused by a combination of
high VWF but also increased sensitivity to collagen
and ADP [73,77,78]. It is therefore not surprising
that the incidence of aspirin non-responders is
reportedly much higher with the PFA-100® than
with other tests [79,80]. The question remains
whether or not this group of patients are at
increased risk for thrombosis. Preliminary data
suggests that PFA-100® CEPI CTs were non-informa-
tive in patients with stable coronary artery disease,
in contrast to LTA [81–84]. However, another study
suggests that the PFA-100® could be informative,
[85] and that shortened CTs with the CADP cartridge
(which is not affected by aspirin) may also be
predictive [77,86-88].
The VerifyNow® Aspirin assay offers the possibil-
ity of rapid and reliable identification of aspirin
resistance or non-responsiveness without the re-
quirement of a specialized laboratory. Indeed, the
test has United States Food and Drug Administration
(FDA) approval for monitoring aspirin therapy and is
being used by some cardiologists and general
practitioners in the USA. The original VerifyNow®
Aspirin cartridge contains fibrinogen-coated beads
and a platelet activator (metallic cations and propyl
gallate) to stimulate the COX-1 pathway and
activate platelets [89]. Ideally, the test should
produce similar results to those obtained by
arachidonic acid-induced LTA. One study showed
an 87% agreement with epinephrine-induced LTA
[90]. Previous data comparing propyl gallate and
other agonists by platelet aggregometry suggest
that this agonist detects a lower number of
responders in volunteers receiving either 100 or
400 mg of aspirin [89]. A more recent study
compared LTA with VerifyNow® Aspirin and PFA-
100® and demonstrated that aspirin non-respon-
siveness was not only higher in both POC tests but
that agreement between the tests was poor and few
patients were non-responsive by all 3 tests [79].
Nevertheless, the VerifyNow® Aspirin test can
330
potentially identify a correlation between aspirin
non-responders, adverse clinical outcomes and
aspirin dose [91–94]. Since the end of 2004 the
VerifyNow® Aspirin cartridge has been modified and
arachidonic acid has replaced propyl gallate as the
principle agonist. Further studies are therefore
warranted to relate adverse clinical outcomes to
the new VerifyNow® Aspirin assay and to see
whether changing therapy based upon the result
can also improve outcomes. A recent study suggests
that aspirin resistant patients, as defined using = 2
out of 3 tests (VerifyNow®Aspirin Assay, LTA with
ADP, LTA with arachidonic acid), also exhibit
reduced responsiveness to clopidogrel, suggesting
that alternative antiplatelet drugs may not be
effective in aspirin resistant patients [58]. In
apparent contrast, we have recently reported that
clopidogrel, in addition to its direct antiplatelet
effect via the P2Y12 ADP receptor, may decrease
aspirin resistance (as defined by a residual arachi-
donic acid-induced increase in platelet surface P-
selectin) in both the presence and absence of
coronary artery disease [64].
Because aspirin inhibits COX-1, measurement of
TXA2 and its metabolites either within serum or
urine provides a relatively simple potential way to
monitor aspirin therapy. In vivo, TXA2 is rapidly
converted into the more stable and inert metabolite
TXB2 which is further metabolized to 11-dehydro
TXB2, the major product found in urine. Measure-
ment of TXB2 by various immunoassays can facilitate
an indirect assessment of the capacity of platelets
to form TXA2. Assays can be standardised so that
TXB2 is measured either within serum derived from
whole blood clotted for 30 min at 37°C or in
supernatants derived from PRP or purified platelets
(with standardized platelet counts) activated by
agonists to stimulate COX-1 activity. The metabolite
11-dehydro TXB2 can also be measured within urine
samples and the assay is also commercially available
as the AspirinWorks® test. This assay has the
advantage that it is non-invasive and one large
study suggested that high levels of urinary 11-
dehydro TXB2 are associated with adverse clinical
events [95].
Monitoring of clopidogrel
Clopidogrel (Plavix) is a prodrug that is metabolized
by cytochrome P450 in the liver to an active
metabolite that specifically and irreversibly blocks
the platelet ADP P2Y12 receptor [96]. Platelet
inhibition by clopidogrel is both dose- and time-
dependent and patients are usually give a loading
dose of 300–600 mg and then maintained on 75 mg/
day. The CAPRIE trial showed that clopidogrel
P. Harrison et al.
prevented more thrombotic vascular events than
aspirin (RRR 8.7%) in patients with known athero-
sclerosis [97]. The CURE trial showed aspirin plus
clopidogrel was 20% more effective than aspirin
alone in acute coronary syndromes,[98] but the
MATCH study showed that adding aspirin to clopido-
grel in high-risk patients with recent ischemic
stroke or transient ischemic attack (TIA) was
associated with a non-significant difference in
reducing major vascular events [99]. Combination
therapy is regarded as the gold standard during
percutaneous coronary intervention (PCI) [100].
However, inter-individual variability in platelet
response to clopidogrel has been observed,[101]
and 5–10% of patients still experience acute or
subacute thrombosis after coronary stent implanta-
tion [56,102–104]. The phenomenon of “clopidogrel
resistance” has been estimated to be between 4%
and 30%. The definition of clopidogrel resistance is
even more complex than aspirin resistance because
the physiological degree of inhibition detected by
ADP-induced LTA can vary widely between indivi-
duals, especially as ADP can also activate platelets
via a second receptor, P2Y1, and there is inter-
individual variability of cytochrome P450 activity
[96]. There is an inverse correlation between P450
3A4 activity and platelet aggregation, and other
drugs can either promote or inhibit metabolism to a
certain degree [105]. Pre-existing variability in ADP
responsiveness is also an important variable and
may provide an explanation for response variability
[106]. Many mechanisms of clopidogrel resistance
have been proposed [55].
Laboratory responses to P2Y12 inhibitors are
largely based upon monitoring ADP-stimulated
responses [107]. Platelets are stimulated with ADP
and responses are monitored using either LTA, the
VerifyNow® P2Y12 assay, TEG platelet mapping
system®, Impact, Plateletworks® flow cytometric
analysis of activation-dependent markers (e.g., P-
selectin, PAC-1), or flow cytometric analysis of
intracellular signaling by monitoring the phosphor-
ylation of vasodilator-stimulated phosphoprotein
(VASP) [25,96,107,108]. Ideally responses are mon-
itored pre- and post-drug. LTA using 5 or 20 μM ADP
can be used to arbitrarily classify patients based
upon measuring the change in (delta) aggregation at
baseline and post-drug [109]. Non-responders can
be defined with a delta aggregation of b10%.
Studies have shown that there is considerable
variation in patient response to clopidogrel and up
to 30% of patients may be non-responders. The
largest analysis so far has found 4% of 544 patients to
be hypo-responsive to clopidogrel [101]. More
recent data suggest that a proportion of patients
are probably under-dosed and that a 600 mg loading
Measuring antiplatelet drug effects in the laboratory
dose significantly reduces the number of non-
responders when compared to 300 mg [109–111].
There is still the critical unresolved question as to
whether in vitro lack of responsiveness to clopido-
grel correlates with the an increased incidence of
adverse events.
ADP-induced LTA is probably not very practical to
test on large numbers of clinical samples. Also, as
residual P2Y1 function can potentially widely vary
despite P2Y12 inhibition, this could not only explain
some of the heterogeneity observed with LTA but
suggests that ADP alone may not be specific enough
to measure the effect of clopidogrel and other P2Y12
antagonists [112]. Despite these problems,
Matetzky et al. found evidence that ADP-induced
LTA predicted adverse events [113]. The VerifyNow
instrument was originally designed to overcome the
major limitations of LTA and can be used as a POC
test. The VerifyNow® P2Y12 cartridge has become
available for monitoring clopidogrel and other P2Y12
antagonists. The assay uses prostaglandin (PG) E1 in
addition to ADP to increase intracellular cyclic
adenosine monophosphate (cAMP), theoretically
enhancing the sensitivity and specificity of the test
for ADP-induced activation of platelets via P2Y12
[114,115]. The PGE1 should suppress the activation
of platelets by P2Y1. The VERITAS (The Verify
Thrombosis Risk Assessment) trial will determine if
the VerifyNow® P2Y12 test is a reliable and sensitive
measure for monitoring clopidogrel therapy. The
combination of ADP and PGE1 is also used in the flow
cytometric-based VASP assay (BioCytex, Marseilles,
France) [112,116]. The principle of this assay is to
measure the phosphorylation of VASP, which is
theoretically proportional to the level of inhibition
of the P2Y12 receptor. Comparison of the VASP assay
with LTA shows that the level of inhibition is higher
in the flow cytometry assay, because non-specific
aggregation can occur via ADP stimulation of P2Y1
[112]. Recent data indeed show that the phosphor-
ylation of VASP correlates with inhibition of LTA
[107]. The PFA-100 is considered unsuitable for
monitoring clopidogrel [32,117–119]. Theoretically
the PFA-100® CADP cartridge may be suitable for
monitoring P2Y12 but both collagen activation and
ADP acting through the P2Y1 receptor, along with the
high shear conditions, may be normally sufficient to
largely overcome P2Y12 blockade [96]. A recent
small study provides evidence that the P2Y1 recep-
tor may be an important variable in determining the
CT in response to P2Y12 blockade [120]. There may
be also be a degree of time- and dose-dependence.
It has also been observed that there is synergy with
clopidogrel/aspirin combination therapy, reflected
as prolongation of both CADP and CEPI CTs
[121,122].
331
Assessment of platelet function by a variety of
tests in correlation with clinical outcomes will be
necessary to define responsiveness to clopidogrel
and other P2Y12 antagonists. Preliminary data from
the CREST study (Clopidogrel Resistance and Stent
Thrombosis study) by Gurbel et al. show differences
with VASP, LTA and activated GPIIb–IIIa responsive-
ness to ADP between patients with and without
subacute stent thrombosis (SAT) [123]. Comparing
data from patients with (n = 20) and without SAT
(n = 100) suggests that clopidogrel response variabil-
ity to ADP is significantly associated with an
increased risk of SAT [123]. This, coupled with
other studies on post-discharge and post-PCI events,
suggests that high post-treatment ex vivo reactivity
to ADP may indeed be an important risk factor for
adverse clinical events [113,116,124].
Carefully controlled, large randomized trials will
be required to define an inadequate response to
P2Y12 inhibition for an individual test and to show that
this correlates with adverse clinical events. Without
such data, therapy should not be altered based upon
the results of any of the tests that purport to
determine responsiveness to a P2Y12 antagonist.
The RESISTOR (Research Evaluation to Study Indivi-
duals who Show Thromboxane or P2Y12 Resistance)
trial that is currently underway in 600 PCI patients
may determine if the level of P2Y12 inhibition
correlates with clinical outcome and if changing
therapy in resistant patients improves outcome.
The development and clinical application of
thienopyridines such as clopidogrel has proven
that the P2Y12 receptor is an attractive target for
new drug development. Because thienopyridines
are metabolized to their active derivatives by the
liver, a number of direct antagonists are in
development (e.g., cangrelor and AZD6140) [96].
Some new thienopyridines (e.g., prasugrel) have
also been developed which exhibit superior proper-
ties (e.g., higher efficacy, faster onset and longer
duration of action) compared with clopidogrel [96].
As some of the observed inter-individual heteroge-
neity of clopidogrel responsiveness may be caused
by differences in liver metabolism, it will be
interesting to determine whether the incidence of
non-responsiveness is lower or even eradicated with
these new drugs and whether high post-treatment
reactivity to ADP remains a potential significant
problem.
Monitoring of GPIIb–IIIa antagonists
The identification of the importance of the GPIIb–
IIIa complex in mediating platelet aggregation (i.e.,
the final common pathway of platelet activation)
suggested that this receptor would be an attractive
332
target for antithrombotic therapy. The FDA-ap-
proved GPIIb–IIIa antagonists (abciximab, tirofiban
and eptifibatide) have now become an important
class of antiplatelet agents that are widely used for
the prevention of thrombotic complications in
patients undergoing PCI or presenting with acute
coronary syndromes. Early observations on the
inhibition of thrombus formation within animal
models not only established a strong correlation
between the level of GPIIb–IIIa blockade and the
prevention of thrombus formation but demonstrat-
ed steep dose-response curves [125,126]. It became
rapidly apparent that a certain level of GPIIb–IIIa
inhibition was required for the optimal efficacy of
GPIIb–IIIa antagonists. This strongly suggested that
monitoring of platelet inhibition could be important
in patients treated with these agents. Monitoring
GPIIb–IIIa antagonists can be performed by a variety
of tests including LTA, WBA, flow cytometry, and
radiolabeled antibody binding assays [127]. Howev-
er some of these tests are time-consuming, expen-
sive and are usually performed within specialized
laboratories. Given the widespread clinical use of
these GPIIb–IIIa antagonists in cardiology, there
existed the potential demand for a simple, inex-
pensive and rapid method that could be utilized as a
POC test either at the bedside or in the clinic, so
that the degree of GPIIb–IIIa blockade could be also
be potentially determined by non-specialists. The
VerifyNow® system was originally developed to
meet this demand. The assay principle was devel-
oped based upon experiments using fibrinogen-
coated beads and TRAP which facilitated the rapid
visual analysis of the degree of GPIIb–IIIa blockade
[20]. The basis of the VerifyNow® IIb/IIIa assay is
that fibrinogen-coated beads will agglutinate in
whole blood in direct proportion to the degree of
platelet activation reflected by exposure of the
fibrinogen binding site on GPIIb–IIIa [18]. The
presence of a GPIIb–IIIa antagonist will therefore
decrease the amount of agglutination in proportion
to the level of inhibition achieved.
Initial in vitro evaluations of the VerifyNow® IIb/
IIIa assay demonstrated good correlations with both
LTA in PRP and radiolabeled receptor binding assays
[18]. Studies in patients receiving abciximab or
other GPIIb–IIIa antagonists also demonstrated good
correlations with LTA [128,129]. GOLD (AU — Asses-
sing Ultegra), a large prospective multicenter study,
showed a significant association between the level
of platelet inhibition by the VerifyNow® IIb/IIIa
assay and clinical outcomes [130]. This suggests that
the device has clinical utility, although no study has
yet been performed to determine whether titration
of GPIIb–IIIa therapy based upon the VerifyNow® IIb/
IIIa test result decreases adverse events.
P. Harrison et al.
A slightly modified Plateletworks® POC assay was
recently reported to correlate more strongly than
VerifyNow® IIb/IIIa with LTA in measuring platelet
inhibition by GPIIb–IIIa antagonists [26]. The PFA-100
has also been utilized to monitor GPIIb–IIIa blockade
and correlates well with LTA and receptor occupancy
measurements [131–133]. Although many patients
give non-closure or N 300 s CT in the PFA-100 following
GPIIb–IIIa antagonist treatment, one study suggests
that failure to observe non-closure may be associated
with an increased risk of cardiac events [133].
Conclusions
As summarized in this review article many tests of
platelet function are now available for clinical use,
and some of these tests have been shown to predict
clinical outcomes after antiplatelet therapy. How-
ever, in most of these studies, the number of major
adverse clinical events was low, and additional
studies are therefore needed. Most importantly, no
published studies address the clinical effectiveness
of altering therapy based on the results of monitor-
ing antiplatelet therapy. Therefore: 1) the correct
treatment, if any, of “resistance” to antiplatelet
therapy is unknown; 2) other than in research trials,
it is not currently appropriate to monitor antiplate-
let therapy in patients or to change therapy based on
such tests [55–57]. A clinically meaningful definition
of “resistance” to antiplatelet drugs needs to be
developed, based on data linking drug-dependent
laboratory tests to clinical outcomes in patients.
Nevertheless, the 2006 American College of Cardi-
ology/American Heart Association guidelines for PCI
provided a Class IIB recommendation (based on level
C evidence) that, in patients in whom subacute stent
thrombosis may be catastrophic or lethal, platelet
aggregation studies may be considered and the
maintenance dose of clopidogrel increased from
75 mg to 150 mg per day if less than 50% inhibition of
platelet aggregation is demonstrated [134].
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